Superfund sites contain complex mixtures of organic and inorganic chemical wastes which can adversely impact environmental and human health. Potential risks associated with these toxic chemicals depends on exposure and hazard/risk assessment of Superfund chemical wastes also requires knowledge of the toxicity of individual chemicals/chemical mixtures and their mechanisms of action. Endocrine disrupters (EDs) are a structurally-diverse class of environmental contaminants that act through disruption of endogenous endocrine signaling pathways, and estrogenic EDs (xenoestrogens) are of particular concern. The overall hypothesis of this project is that xenoestrogen-mediated ER signaling is highly complex and dependent on ligand structure, nuclear ER complex/promoter interactions, non-genomic pathways, and tissue specificity. Therefore, the proposed studies will investigate activation of several key genomic/non-genomic estrogenic pathways by E2, diethylstilbestrol, structurally diverse xenoestrogens (organic and metallic), selected phytoestrogens, and other well-characterized selective ER modulators (SERMs). Aim 1 will focus on the genomic action of xenoestrogen-dependent activation of ERalpha and ERalpha/Sp1 -mediated gene expression using in vitro and in vivo models. Several metal salts, including cadmium chloride, exhibit estrogenic activity, and Aim 2 will determine their comparative activation of ERE and GC-rich promoters using in vitro and in'vivo models comparable to those outlined in Aim 1. The mitogenic activity of E2 is also due to rapid activation of several kinase-dependent phosphorylation pathways that are dependent on extranuclear ER. Several assays have now been developed for detecting activation of protein kinase A, protein kinase C, calmodulin-dependent kinase IV, mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways by E2 in breast cancer cells. Aim 3 will investigate structure-dependent activation of kinases in different cell lines by xenoestrogens and related ER ligands and determine the potential role of non-genomic pathways in their estrogenic activity. The mechanistic similarities and differences in the estrogenic activity of xenoestrogens, phytoestrogens, and related compounds determined in this project will augment results of more traditional receptor binding and transactivation assays and provide a more rationale basis for hazard and risk assessment of estrogenic EDs.